Resumo Introdução: Citomegalovírus (CMV) é um dos agentes infecciosos mais comuns em pacientes com transplante de órgãos sólidos, com morbidade e mortalidade significativas. Objetivo: Este estudo visou estabelecer um limite para o início do tratamento preemptivo. Além disso, comparou o desempenho da antigenemia com os resultados da qPCR in house. Desenho do estudo: Este foi um estudo de coorte prospectivo realizado em 2017 em um centro único de transplante renal no Brasil. A validação clínica foi realizada comparando resultados de qPCR in house, com o padrão de atendimento na época (Antigenemia para CMV Pp65). A análise da curva ROC foi realizada para determinar o limite ideal para o início da terapia preemptiva baseado nos resultados do teste qPCR in house. Resultados: 232 amostras de 30 pacientes foram testadas com antigenemia e qPCR, das quais 163 (70,26%) foram concordantes (Coeficiente Kappa: 0,435, p<0,001; Correlação Spearman: 0,663). PCR permitiu diagnósticos precoces. O número médio de dias para o primeiro resultado positivo foi 50 (intervalo, 24-105) para antigenemia e 42 (intervalo, 24-74) para qPCR (p<0,001). A análise da curva ROC revelou que em um limite de 3.430 UI/mL (Log 3,54), qPCR teve sensibilidade de 97,06% e especificidade de 74,24% (AUC 0,92617 ± 0,0185, p<0,001), na previsão de 10 células/10(5) leucócitos por antigenemia e na decisão do médico de tratar. Conclusões: Antigenemia para CMV Pp65 e qPCR para CMV mostraram uma concordância aceitável e uma correlação moderada neste estudo. qPCR in house revelou-se um método preciso para determinar DNAemia do CMV em pacientes transplantados renais, obtendo resultados positivos semanas antes da antigenemia.

Abstract Introduction: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. Objective: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. Study design: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. Results: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. Conclusions: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

ABSTRACT Introduction: Cytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. Objective: This study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. Study design: The methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n = 30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. Results: The in-house assay had a limit of detection and quantification of 60.3 IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R 2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. Conclusions: These results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Abstract We present postmortem evidence of invasive pulmonary aspergillosis (IPA) in a patient with severe COVID-19. Autopsies of COVID-19 confirmed cases were performed. The patient died despite antimicrobials, mechanical ventilation, and vasopressor support. Histopathology and peripheral blood galactomannan antigen testing confirmed IPA. Aspergillus penicillioides infection was confirmed by nucleotide sequencing and BLAST analysis. Further reports are needed to assess the occurrence and frequency of IPA in SARS-CoV-2 infections, and how they interact clinically.

ABSTRACT The Choosing Wisely Initiative aims to collect statements from medical societies all over the world on medical interventions that result in no benefit to patients, with the potential to cause harm. In this article we present the views of the Diagnostic Laboratory Group at the Brazilian Society of Infectious Diseases (SBI). Ten experts from SBI were asked to list 10 diagnostic tests that were perceived as unnecessary in the field of infectious diseases. After voting for the more relevant topics, a questionnaire was sent to all SBI members, in order to select for the most important items. A total of 482 votes were obtained, and the top 10 results are shown in this manuscript. The Choosing Wisely statements of SBI should facilitate clinical practice by optimizing the use of diagnostic resources in the field of infectious diseases.

ABSTRACT Introduction Clostridium difficile is an important cause of diarrhoea, particularly in patients receiving antibiotic therapy. Recent studies have shown that a substantial proportion of C. difficile infections are acquired in the community, as a zoonotic disease. Brazil is a large exporter of meat and so far no study has evaluated meat contamination with C. difficile spores. Methods Here we analysed 80 retail meat products purchased from local supermarkets in a Brazilian metropolis (Porto Alegre, Southern Brazil). Samples from these products were grown in anaerobic conditions, and tested with a real time polymerase chain reaction test. Results Contamination with C. difficile spores was not found in the study. Bacteria isolated from meat included Streptococcus gallolyticus, Lactobacillus plantarum, Enterococcus gallinarum and Pediococcus acidilactici. Discussion Close vigilance is required in order to guarantee the quality of Brazilian retail meat in the long term.

Resumo Introdução: A infecção pelo vírus BK (BKV) em pacientes de transplante renal pode levar a disfunção do aloenxerto renal e perda do enxerto. A determinação precisa da carga viral do BKV é fundamental para prevenir a nefropatia associada ao BKV (BKVAN), mas o ponto de corte de melhor valor preditivo para BKVAN ainda é foco de debates. Objetivo: Avaliar o desempenho de um teste de qPCR comercial e outro desenvolvido internamente para detecção quantitativa de vírus BK em receptores de transplante renal. Métodos: O presente estudo prospectivo incluiu receptores de transplante renal de dois grandes hospitais universitários no Brasil. Os pacientes foram testados para infecção por BKV a cada três meses no primeiro ano pós-transplante com um teste comercial de reação em cadeia de polimerase quantitativa em tempo real (qPCR) e outro desenvolvido internamente. A presença de BKVAN foi confirmada com base na histopatologia. A área sob a curva para o qPCR plasmático foi determinada a partir da análise da característica de operação do receptor. Resultados: Um total de 200 pacientes foram incluídos. Cinquenta e oito por cento eram do sexo masculino, 19,5% tinham diabetes mellitus e 82% tiveram seus rins transplantados de doadores falecidos. Viremia de BKV foi detectada em 32,5% dos pacientes e oito (4%) foram diagnosticados com BKVAN. BKVAN foi associada a viremia de 4,1 log cópias/mL usando o kit comercial. O corte para o ensaio interno foi de 6,1 log cópias/mL. A linearidade entre o kit comercial e o ensaio interno foi R2 = 0,83. Conclusão: Nosso estudo demonstrou uma acentuada variabilidade na carga viral de BKV quando diferentes metodologias de qPCR foram utilizadas. O ensaio interno de qPCR mostrou-se clinicamente útil, além de ser uma opção menos onerosa em relação aos kits comerciais de qPCR. Há uma necessidade urgente de se definir padrões de BKV para a comunidade internacional.

Abstract Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

ABSTRACT Introduction: Invasive aspergillosis is a condition associated with a high mortality rate mostly due to difficulties in performing an early diagnosis. In recent years, galactomannan detection has markedly improved the diagnosis of invasive aspergillosis, but very little is known on how physicians deal with this test in clinical practice. Methods: This cross-sectional study aimed to analyze the indications for the use of serum galactomannan in a large Brazilian hospital, between 2015 and 2016. No specific protocol was in place for GM request. We reviewed the medical records of adult (>18 years-old) patients who were tested for galactomannan due to one the following indications: screening, diagnosis, or treatment follow-up. Additional variables included demographic data, underlying diseases, presence of neutropenia, and use of previous antifungal (anti-Aspergillus) drugs. Results: The mean age of the patients was 51 years-old (sd ± 15.8), and 63.3% of patients were male. Patients with hematological malignancies accounted for 60.1% of the cases, mostly acute myeloid leukemia (19.6%). Galactomannan testing was positive in 12.2% of patients, including 1.6% of occasions in which the test was used for screening purposes, 13.2% for diagnosis, and 32.4% during follow-up. Median time for chest imaging request was two days before GM testing. Previous antifungal therapy was reported for 35.1% of patients, mostly amphotericin B (57.1%). Conclusion: The correct use of galactomannan testing is essential for an early diagnosis of invasive aspergillosis, which may improve the prognosis of the disease. We demonstrated that clinicians usually ask for galactomannan tests to confirm imaging findings in patients who frequently were on antifungal drugs, something that could be improved by medical education. We observed a low frequency of galactomannan use for preemptive antifungal therapy (25.7%), which is worrying considering the well-known beneficial use of GM testing in this scenario.

Abstract The diagnosis of progressive disseminated histoplasmosis is often a challenge to clinicians, especially due to the low sensitivity and long turnaround time of the classic diagnostic methods. In recent years, studies involving a variety of non-culture-based diagnostic tests have been published in the literature. We performed a systematic review by selecting studies evaluating non-culture-based diagnostic methods for progressive disseminated histoplasmosis. We searched for articles evaluating detection of antibody, antigens, as well as DNA-based diagnostic methods. A comprehensive PUBMED, Web of Science, and Cochrane Library search was performed between the years 1956 and 2016. Case reports, review articles, non-human models and series involving less than 10 patients were excluded. We found 278 articles and after initial review 18 articles were included: (12) involved antigen detection methods, (4) molecular methods, and (2) antibody detection methods. Here we demonstrate that the pursuit of new technologies is ultimately required for the early and accurate diagnosis of disseminated histoplasmosis. In particular, urinary antigen detection was the most accurate tool when compared with other diagnostic techniques.

Resumo A citologia urinária e a reação da cadeia da polimerase em tempo real (qPCR) em amostras de sangue e/ou urina são comumente utilizados para rastrear nefropatia associada ao polyomavirus (PVAN), em pacientes transplantados renais. Entretanto, poucos estudos comparam diretamente esses testes diagnósticos quanto ao desempenho para predizer esta complicação. Aqui realizamos uma revisão sistemática na qual foram estudados pacientes transplantados renais adultos (≥ 18 anos). Uma pesquisa estruturada Pubmed foi utilizada para identificar estudos comparando citologia urinária e/ou qPCR em amostras de urina e plasma para detectar PVAN, utilizando a biópsia renal como padrão-ouro para o diagnóstico. Dentre os 707 artigos em potencial, apenas 12 atendiam aos critérios de inclusão e foram analisados em maior detalhe. Foram incluídos 1694 pacientes transplantados renais, entre os quais 115 (6,8%) classificados com PVAN presuntivo e 57 (3,4%) PVAN confirmado. Nessa revisão sistemática, o qPCR no plasma tive melhor desempenho para PVAN em comparação com citopatologia urinária.

Abstract Urine cytology and qPCR in blood and urine are commonly used to screen renal transplant recipients for polyomavirus-associated nephropathy (PVAN). Few studies, however, have directly compared these two diagnostic tests, in terms of their performance to predict PVAN. This was a systematic review in which adult (≥ 18 years old) renal transplant recipients were studied. A structured Pubmed search was used to identify studies comparing urine cytology and/or qPCR in urine and plasma samples for detecting PVAN with renal biopsy as the gold standard for diagnosis. From 707 potential papers, there were only twelve articles that matched the inclusion criteria and were analyzed in detail. Among 1694 renal transplant recipients that were included in the review, there were 115 (6.8%) patients with presumptive PVAN and 57 (3.4%) PVAN confirmed. In this systematic review, the qPCR in plasma had better performance for PVAN compared to urine cytopathology.

OBJETIVO: Identificar preditores clínicos, laboratoriais e radiológicos da infecção por Bordetella pertussis.MÉTODOS: Trabalho retrospectivo, com análise de prontuários clínicos de todos os indivíduos submetidos ao diagnóstico molecular (qPCR) para B. pertussis de setembro de 2011 à janeiro de 2013. Foram revistos dados clínicos e laboratoriais, incluindo informações sobre idade, sexo, sinais/sintomas, tempo de hospitalização, contagens de células sanguíneas, exames de imagem, co-infecção com outros patógenos respiratórios, e evolução clínica.RESULTADOS: 222 casos foram revistos, do quais 72,5% tinham coqueluche confirmada, sendo 60,9% menores de um ano de idade. Foram observados preditores independentes para B. pertussis em pacientes com menos de seis meses de idade. Nesses casos, os preditores identificados foram cianose (OR 8,0; CI 95% 1,8-36,3; p=0,007) e contagem de linfócitos >104/µL (OR 10,0, CI 95% 1,8-54,5; p=0,008). Preditores de coqueluche não puderam ser determinados para crianças maiores de 6 meses de idade. Coinfecção foi encontrada em 21,4% dos pacientes, dos quais 72,7% tinham até seis meses de idade, sendo que o adenovírus foi o agente mais comum (40,9%). Nesses indivíduos, não foram observadas características clíncias capazes de distinguir pacientes com co-infecção, porém foi verificado um maior tempo de internação hospitalar nos pacientes com mais de um agente infeccioso detectado (12 vs. 6 dias; p=0,009).CONCLUSÕES: Cianose e linfocitose são preditores independentes para coqueluche em crianças com até seis meses de idade.

OBJECTIVE: To identify clinical, laboratorial and radiographic predictors for Bordetella pertussis infection.METHODS: This was a retrospective study, which analyzed medical records of all patients submitted to a molecular dignosis (qPCR) for B. pertussis from September 2011 to January 2013. Clinical and laboratorial data were reviewed, including information about age, sex, signs/symptoms, length of hospitalization, blood cell counts, imaging findings, coinfection with other respiratory pathogens and clinical outcome.RESULTS: 222 cases were revised. Of these, 72.5% had proven pertussis, and 60.9% were under 1 year old. In patients aging up to six months, independent predictors for B. pertussisinfection were (OR 8.0, CI 95% 1.8-36.3; p=0.007) and lymphocyte count >104/µL (OR 10.0, CI 95% 1.8-54.5; p=0.008). No independent predictors of B. pertussisinfection could be determined for patients older than six months. Co-infection was found in 21.4% of patients, of which 72.7% were up to six months of age. Adenovirus was the most common agent (40.9%). In these patients, we were not able to identify any clinical features to detect patients presenting with a respiratory co-infection, even though longer hospital stay was observed in patients with co-infections (12 vs. 6 days; p=0.009).CONCLUSIONS: Cyanosis and lymphocytosis are independent predictors for pertussis in children up to 6 months old.

Um método simples, sensível e seletivo para determinação de voriconazol em plasma e fluido oral empregando cromatografia líquida de ultra-eficiência foi desenvolvido e validado. Após extração líquido-líquido do plasma e fluido oral com metil-tert-butil éter, o analito e o padrão interno foram separados numa coluna Hypersil Gold C18 (2,1 100 mm, d.p. 1,9 µm), eluída isocraticamente com uma mistura de tampão fosfato trietilamônio pH 3,0 e acetonitrila (70:30, v/v). O tempo total da análise foi de 4 min, com consumo total de fase móvel de 2,2 mL. A determinação foi realizada com detector de arranjo de fotodiodos com quantificação em 256 nm. As concentrações de voriconazol no fluido oral foram, em média, 57,5% (± 5,3) daquelas determinadas em amostras pareadas de plasma.

A simple, sensitive and selective ultra-performance liquid chromatography method for the determination of voriconazole in plasma and oral fluid was developed and validated. After a liquid-liquid extraction with methyl-tert-butyl ether, the analyte and internal standard were separated on a Hypersil Gold C18 column (2.1 × 100 mm, p.d. 1.9 µm), eluted with a mobile phase composed of thietylammonium phosphate buffer and acetonitrile (70:30, v/v). Total run time was 4 min, total mobile phase consumption of 2.2 mL. Detection was performed with a photodiode array detector with quantitation at 256 nm. Voriconazole concentrations in oral fluid were on average 57.5% (±5.3) of those measured in paired plasma samples.

The best strategy for control of cytomegalovirus (CMV) infection in lung transplant patients is still not determined. The aim of this study was to document the incidence of CMV infection in a cohort of lung transplant recipients under universal prophylaxis with intravenous ganciclovir. All patients received immunosuppressive regimens consisting of cyclosporine, azathioprine, and prednisone. Regardless of CMV serostatus, intravenous ganciclovir was prescribed for every patient in the first 3 months post-transplantation. CMV infection was defined as the detection of CMV pp65 in leukocytes. Eighty-two lung transplant patients were included over a 5-year period. The incidence of CMV infection in the first year post-transplantation was 68.3%, occurring after a median length of 114 days (range, 26-343 days). This study revealed a high incidence of CMV infection in the first year following lung transplantation despite prolonged universal ganciclovir prophylaxis.